Electrochemical test strip kit for analyte determination

ABSTRACT

Electrochemical test strips and methods for their use in the detection of an analyte in a physiological sample are provided. The subject test strips have a reaction zone defined by opposing metal electrodes separated by a thin spacer layer. The metal surface of at least one of the electrodes is modified by a homogenous surface modification layer made up of linear self-assembling molecules having a first sulfhydryl end group and a second sulfonate end group separated by a short chain alkyl linking group, where 2-mercaptoethane sulfonic acid or a salt thereof is preferred in certain embodiments. The subject electrochemical test strips find application in the detection of a wide variety of analytes, and are particularly suited for use the detection of glucose.

CROSS-REFERENCES

This application is a divisional of U.S. patent application Ser. No.09/497,269, filed Feb. 2, 2000, which is herein incorporated byreference.

INTRODUCTION

1. Field of the Invention

The field of this invention is analyte determination, particularlyelectrochemical analyte determination and more particularly theelectrochemical determination of blood analytes.

2. Background

Analyte detection in physiological fluids, e.g. blood or blood derivedproducts, is of ever increasing importance to today's society. Analytedetection assays find use in a variety of applications, includingclinical laboratory testing, home testing, etc., where the results ofsuch testing play a prominent role in diagnosis and management in avariety of disease conditions. Analytes of interest include glucose fordiabetes management, cholesterol, and the like. In response to thisgrowing importance of analyte detection, a variety of analyte detectionprotocols and devices for both clinical and home use have beendeveloped.

One type of method that is employed for analyte detection is anelectrochemical method. In such methods, an aqueous liquid sample isplaced into a reaction zone in an electrochemical cell comprising twoelectrodes, i.e. a reference and working electrode, where the electrodeshave an impedance which renders them suitable for amperometricmeasurement. The component to be analyzed is allowed to react directlywith an electrode, or directly or indirectly with a redox reagent toform an oxidizable (or reducible) substance in an amount correspondingto the concentration of the component to be analysed, i.e. analyte. Thequantity of the oxidizable (or reducible) substance present is thenestimated electrochemically and related to the amount of analyte presentin the initial sample.

In electrochemical analyte detectors used to practice the abovedescribed methods, it is often desirable to modify the surface of themetal electrodes to be hydrophilic. A variety of different techniqueshave been developed to modify the surfaces of metal electrodes. However,such surface modified electrodes tend to have limited storage life, thuslimiting their potential applications.

As such, there is continued interest in the identification of newmethods for modifying metallic electrode surfaces for use in theelectrochemical detection of analytes. Of particular interest would bethe development of a method which resulted in a storage stablehydrophilic surface that provided rapid wicking time and did notinterfere with the electrochemical measurements of the electrode.

Relevant Literature

U.S. Patent documents of interest include: U.S. Pat. Nos. 5,834,224;5,942,102 and 5,972,199. Other patent documents of interest include WO99/49307; WO 97/18465 and GB 2 304 628. Other references of interestinclude: Dalmia et al, J. Electroanalytical Chemistry (1997) 430:205-214; Nakashima et al., J. Chem. Soc. (1990) 12: 845-847; and Palacinet al., Chem. Mater. (1996) 8:1316-1325.

SUMMARY OF THE INVENTION

Electrochemical test strips and methods for their use in the detectionof an analyte, e.g. glucose, in a physiological sample, e.g. blood, areprovided. The subject test strips have a reaction area defined byopposing metal electrodes separated by a thin spacer layer. The metalsurface of at least one of the electrodes is modified by a homogenoussurface modification layer made up of linear self-assembling moleculeshaving a first sulfhydryl end group and a second sulfonate end groupseparated by a short chain alkyl linking group, where 2-mercaptoethanesulfonic acid or a salt thereof is preferred in certain embodiments. Thesubject electrochemical test strips find application in the detection ofa wide variety of analytes, and are particularly suited for use thedetection of glucose.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 and 2 provide a representation of an electrochemical test stripaccording to the subject invention.

FIG. 3 provides an analysis of the contact angle of various cystinetreated metallic electrodes at various times following treatment.

FIG. 4 provides an analysis of the wicking time of various cystinetreated metallic electrodes at various times following treatment.

FIGS. 5A and 5B provide an analysis of the contact angle of various MESAtreated metallic electrodes at various times following treatment.

FIG. 6 provides an analysis of the wicking time of various MESA treatedmetallic electrodes at various times following treatment.

FIG. 7 provides a comparison of the wicking time of various cystine andMESA coated electrodes.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Electrochemical test strips for use in analyte detection in aphysiological sample are provided. In the subject test strips, twoopposing metal electrodes separated by a thin spacer layer define areaction area. A critical feature of the subject test strips is that atleast one of the metal electrodes has a surface that is modified with asurface modification layer made up of linear molecules having asulfhydryl end group and a sulfonate end group separated by a loweralkyl linking group. Present in the reaction area are redox reagentscomprising an enzyme and a mediator. Also provided are methods of usingthe subject test strips in analyte detection, e.g. glucosedetermination. In further describing the subject invention, theelectrochemical test strip will be described first, followed by a morein depth review of the subject methods for using the test strips inanalyte detection.

Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

In this specification and the appended claims, singular referencesinclude the plural, unless the context clearly dictates otherwise.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs.

Electrochemical Test Strips

As summarized above the electrochemical test strips of the subjectinvention are made up of two opposing metal electrodes separated by athin spacer layer, where these components define a reaction area inwhich is located a redox reagent system. A representation of anelectrochemical test strip according to the subject invention isprovided in FIGS. 1 and 2. Specifically, FIG. 1 provides an explodedview of an electrochemical test strip 10 which is made up of workingelectrode 12 and reference electrode 14 separated by spacer layer 16which has a cutaway section 18 that defines the reaction zone or area inthe assembled strip. FIG. 2 shows the same test strip in assembled form.Each of the above elements, i.e. the working and reference electrodes,the spacer layer and the reaction area are now described separately ingreater detail.

Electrodes

As indicated above, the subject electrochemical test strips include aworking electrode and a reference electrode. Generally, the working andreference electrodes are configured in the form of elongated rectangularstrips. Typically, the length of the electrodes ranges from about 1.9 to4.5 cm, usually from about 2 to 2.8 cm. The width of the electrodesranges from about 0.38 to 0.76 cm, usually from about 0.51 to 0.67 cm.The reference electrodes typically have a thickness ranging from about10 to 100 nm and usually from about 18 to 22 nm. In certain embodiments,the length of one of the electrodes is shorter than the length of theother electrode, wherein in certain embodiments it is about 0.32 cmshorter.

The working and reference electrodes are further characterized in thatat least the surface of the electrodes that faces the reaction area inthe strip is a metal, where metals of interest include palladium, gold,platinum, silver, iridium, carbon, doped indium tin oxide, stainlesssteel and the like. In many embodiments, the metal is gold or palladium.

While in principle the entire electrode may be made of the metal, eachof the electrodes is generally made up of an inert support material onthe surface of which is present a thin layer of the metal component ofthe electrode. In these more common embodiments, the thickness of theinert backing material typically ranges from about 51 to 356 μm, usuallyfrom about 10 to 153 μm while the thickness of the metal layer typicallyranges from about 10 to 100 nm and usually from about 20 to 40 nm, e.g.a sputtered metal layer. Any convenient inert backing material may beemployed in the subject electrodes, where typically the material is arigid material that is capable of providing structural support to theelectrode and, in turn, the electrochemical test strip as a whole.Suitable materials that may be employed as the backing substrate includeplastics, e.g. PET, PETG, polyimide, polycarbonate, polystyrene,silicon, ceramic, glass, and the like.

The subject test strips are further characterized in that at least oneof the metallic surfaces of the electrodes, and in some embodiments bothof the metallic surfaces of the electrodes, that face, i.e. border orbound, the reaction area, have a surface modification layer presentthereon. The surface modification layer is a homogenous layer ofself-assembling molecules that renders the surface stably hydrophilic ina storage stable manner. More specifically, the surface modificationlayer should impart to the surface a low contact angle, typicallyranging from about 10 to 30 and usually from about 15 to 25° and a fastwicking time, e.g. 0.5 to 2 and usually from about 1 to 2 s, even afteran extended period of time at an elevated temperature, e.g. even after 7to 14 days at a temperature of from about 4 to 56° C.

By homogenous is meant that the surface modification layer is made up ofthe same type of molecules. In other words, all of the self-assemblingmolecules in the surface modification layer are identical. Generally,the self-assembling molecule that makes up the surface modificationlayer is a linear molecule having a sulfhydryl end group and a sulfonateend group separated by a lower alkyl linking group. The term sulfonateend group is used herein to refer to both a sulfonic acid moiety as wellas a sulfonate moiety, which may be associated with a cation, e.g.sodium, as is found in a sulfonate salt. The alkyl linking groupgenerally ranges from about 1 to 8, usually from about 1 to 6 carbonatoms in length, and may or may not include one or more sites ofunsaturation, but is generally a saturated molecule. In certainembodiments, the number of carbon atoms in the alkyl linking groupranges from about 1 to 4 and often from about 1 to 3, with methylene andethylene linking groups being common in these embodiments.

In many embodiments, the molecule that makes up the self-assemblingsurface modification layer is a molecule of the formula:HS—(CH₂)_(n)—SO₃Y

wherein:

-   -   n is an integer from 1 to 6; and    -   Y is H or a cation.

Of particular interest in many embodiments of the subject invention aresurface modification layers made up of 2-mercaptoethane ethane sulfonicacid or a salt thereof, e.g. 2-mercaptoethane sulfonate sodium.

The working and reference electrodes as described above may befabricated using any convenient protocol. A representative protocolincludes preparation of the metal electrodes by first sputtering themetal layer of sufficient thickness onto the surface of the inertbacking material. Next, the electrode(s) to be surface modified, or atleast the metallic surface that is to be modified, to have the surfacemodification layer is contacted with a fluid composition, e.g. anaqueous organic solution, of the self-assembling molecule. Contact maybe achieved by any convenient means, including submersion slot coating,grevure printing of the electrode into the composition. Theconcentration of the self-assembling molecule in the fluid compositiontypically ranges from about 0.5 to 1%, usually from about 0.05 to 0.5%and more usually from about 0.05 to 0.3%. Contact is maintained for asufficient period of time for the monolayer to form, e.g. for a periodof time ranging from about 0.5 to 3 minutes, usually from about 0.5 to 2min, followed by drying of the electrode surface for use in the subjectelectrochemical test strips. A more detailed representative fabricationprofile is provided in the experimental section, infra.

Spacer Layer

A feature of the subject electrochemical test strips is that the workingand reference electrodes as described above face each other and areseparated by only a short distance, such that the distance between theworking and reference electrode in the reaction zone or area of theelectrochemical test strip is extremely small. This minimal spacing ofthe working and reference electrodes in the subject test strips is aresult of the presence of a thin spacer layer positioned or sandwichedbetween the working and reference electrodes. The thickness of thisspacer layer generally ranges from about 1 to 500 um, usually from about102 to 153 um. The spacer layer is cut so as to provide a reaction zoneor area with at least an inlet port into the reaction zone, andgenerally an outlet port out of the reaction zone as well. Arepresentative spacer layer configuration can be seen in FIGS. 1 and 2.While the spacer layer is shown in these figures as having a circularreaction area cut with side inlet and outlet vents or ports, otherconfigurations are possible, e.g. square, triangular, rectangular,irregular shaped reaction areas, etc. The spacer layer may be fabricatedfrom any convenient material, where representative suitable materialsinclude PET, PETG, polyimide, polycarbonate and the like, where thesurfaces of the spacer layer may be treated so as to be adhesive withrespect to their respective electrodes and thereby maintain thestructure of the electrochemical test strip. Of particular interest isthe use of a die-cut double-sided adhesive strip as the spacer layer.

Reaction Zone

The subject electrochemical test strips include a reaction zone or areathat is defined by the working electrode, the reference electrode andthe spacer layer, where these elements are described above.Specifically, the working and reference electrodes define the top andbottom of the reaction area, while the spacer layer defines the walls ofthe reaction area. The volume of the reaction area is at least about 0.1μl, usually at least about 1 μl and more usually at least about 1.5 μl,where the volume may be as large as 10 μl or larger. As mentioned above,the reaction area generally includes at least an inlet port, and in manyembodiments also includes an outlet port. The cross-sectional area ofthe inlet and outlet ports may vary as long as it is sufficiently largeto provide an effective entrance or exit of fluid from the reactionarea, but generally ranges from about 9×10⁻⁵ to 5×10⁻³ cm², usually fromabout 1.3×10⁻³ to 2.5×10⁻³ cm².

Present in the reaction area is a redox reagent system, which reagentsystem provides for the species that is detected by the electrode andtherefore is used to derive the concentration of analyte in aphysiological sample. The redox reagent system present in the reactionarea typically includes at least an enzyme(s) and a mediator. In manyembodiments, the enzyme member(s) of the redox reagent system is anenzyme or plurality of enzymes that work in concert to oxidize theanalyte of interest. In other words, the enzyme component of the redoxreagent system is made up of a single analyte oxidizing enzyme or acollection of two or more enzymes that work in concert to oxidize theanalyte of interest. Enzymes of interest include oxidases,dehydrogenases, lipases, kinases, diaphorases, quinoproteins and thelike.

The specific enzyme present in the reaction area depends on theparticular analyte for which the electrochemical test strip is designedto detect, where representative enzymes include: glucose oxidase,glucose dehydrogenase, cholesterol esterase, cholesterol oxidase,lipoprotein lipase, glycerol kinase, glycerol-3-phosphate oxidase,lactate oxidase, lactate dehydrogenase, pyruvate oxidase, alcoholoxidase, bilirubin oxidase, uricase, and the like. In many preferredembodiments where the analyte of interest is glucose, the enzymecomponent of the redox reagent system is a glucose oxidizing enzyme,e.g. a glucose oxidase or glucose dehydrogenase.

The second component of the redox reagent system is a mediatorcomponent, which is made up of one or more mediator agents. A variety ofdifferent mediator agents are known in the art and include:ferricyanide, phenazine ethosulphate, phenazine methosulfate,pheylenediamine, 1-methoxy-phenazine methosulfate,2,6-dimethyl-1,4-benzoquinone, 2,5-dichloro-1,4-benzoquinone, ferrocenederivatives, osmium bipyridyl complexes, ruthenium complexes and thelike. In those embodiments where glucose in the analyte of interest andglucose oxidase or glucose dehydrogenase are the enzyme components,mediator of particular interest is ferricyanide. Other reagents that maybe present in the reaction area include buffering agents, e.g.citraconate, citrate, phosphate, “Good” buffers and the like.

The redox reagent system is generally present in dry form. The amountsof the various components may vary, where the amount of enzyme componenttypically ranges from about 0.1 to 10% by weight.

Methods

Also provided by the subject invention are methods of using the subjectelectrochemical test strips to determine the concentration of an analytein a physiological sample. A variety of different analytes may bedetected using the subject test strips, where representative analytesinclude glucose, cholesterol, lactate, alcohol, and the like. In manypreferred embodiments, the subject methods are employed to determine theglucose concentration in a physiological sample. While in principle thesubject methods may be used to determine the concentration of an analytein a variety of different physiological samples, such as urine, tears,saliva, and the like, they are particularly suited for use indetermining the concentration of an analyte in blood or blood fractions,and more particularly in whole blood.

In practicing the subject methods, the first step is to introduce aquantity of the physiological sample into the reaction area of the teststrip, where the electrochemical test strip is described supra. Theamount of physiological sample, e.g. blood, that is introduced into thereaction area of the test strip may vary, but generally ranges fromabout 0.1 to 10 ul, usually from about 1 to 1.6 ul. The sample may beintroduced into the reaction area using any convenient protocol, wherethe sample may be injected into the reaction area, allowed to wick intothe reaction area, and the like, as may be convenient.

Following application of the sample to the reaction zone, anelectrochemical measurement is made using the reference and workingelectrodes. The electrochemical measurement that is made may varydepending on the particular nature of the assay and the device withwhich the electrochemical test strip is employed, e.g. depending onwhether the assay is coulometric, amperometric or potentiometric.Generally, the electrochemical measure will measure charge(coulometric), current (amperometric) or potential (potentiometric),usually over a give period of time following sample introduction intothe reaction area. Methods for making the above describedelectrochemical measurement are further described in U.S. Pat. Nos.4,224,125; 4,545,382; and 5,266,179; as well as WO 97/18465; WO99/49307; the disclosures of which are herein incorporated by reference.

Following detection of the electrochemical signal generated in thereaction zone as described above, the amount of the analyte present inthe sample introduced into the reaction zone is then determined byrelating the electrochemical signal to the amount of analyte in thesample. In making this derivation, the measured electrochemical signalis typically compared to the signal generated from a series ofpreviously obtained control or standard values, and determined from thiscomparison. In many embodiments, the electrochemical signal measurementsteps and analyte concentration derivation steps, as described above,are performed automatically by a devices designed to work with the teststrip to produce a value of analyte concentration in a sample applied tothe test strip. A representative reading device for automaticallypracticing these steps, such that user need only apply sample to thereaction zone and then read the final analyte concentration result fromthe device, is further described in copending U.S. application Ser. No.09/333,793, entitled “Sample Detection to Initiate Timing of anElectrochemical Assay,” the disclosure of which is herein incorporatedby reference.

Kits

Also provided by the subject invention are kits for use in practicingthe subject methods. The kits of the subject invention at least includean electrochemical test strip with at least one surface modified metalelectrode, as described above. The subject kits may further include ameans for obtaining a physiological sample. For example, where thephysiological sample is blood, the subject kits may further include ameans for obtaining a blood sample, such as a lance for sticking afinger, a lance actuation means, and the like. In addition, the subjectkits may include a control solution, e.g. a glucose control solutionthat contains a standardized concentration of glucose. In certainembodiments, the kits also comprise an automated instrument, asdescribed above, for detecting an electrochemical signal using theelectrodes following sample application and relating the detected signalto the amount of analyte in the sample. Finally, the kits includeinstructions for using the subject reagent test strips in thedetermination of an analyte concentration in a physiological sample.These instructions may be present on one or more of the packaging, alabel insert, containers present in the kits, and the like.

The following examples are offered by way of illustration and not by wayof limitation.

Experimental

I. Preparation of Electrochemical Test Strips

A. Preparation of MESA Treated Electrochemical Test Strips

A (0.1)1% 2-mercaptoethane sulfonic acid (MESA) solution is prepared bydissolving 1.000 gm MESA (TCI, Catalog # M0913) into 999 gm Milli Qwater. Gold and palladium sheets are prepared by sputtering the surfaceof a 7 mil thick polyester substrate with gold or palladium such that asurface metallic layer of 100 to 500 angstroms is obtained. Followingpreparation of these gold and palladium master rolls, 12 in×8.5 inchsheets are cut. The sheets are then immersed in the 1% MESA solution for1 minute. The coated sheet is then air dried for 1 hour and tested forcontact angle using a Goniometer and water as described in Procedure Afound in Appendix A, infra, to ensure that the contact angle is <20°.

Test strips having dimensions of 0.2×1.2 inch are then cut from theabove gold and metal sheets and are used to fabricate electrochemicaltest strips as follows. A gold strip and palladium strip are used tosandwich a die-cut double sided pressure sensitive adhesive strip havinga thickness of 0.005 in and a circular die-cut area that defines thereaction zone, inlet and outlet ports when sandwiched between the goldand metal strips, as shown in FIGS. 1 and 2. A dry reagent consisting ofbuffer, mediator, enzyme and bulking agents is ink jetted onto thepalladium electrode prior to sandwiching the double-sided adhesive.

B. Preparation of Cystine Treated Electrochemical Test Strips

Cystine treated electrochemical test strips were prepared according to astandard industry protocol.

II. Characterization of Cystine Treated Electrochemical Test Strips

A. Contact Angle

The contact angle of cystine treated gold and palladium test strips wasdetermined with water and a goniometer as described in Procedure B foundin Appendix A, infra. The contact angle was determined at various timesfollowing surface treatment, i.e. 0, 7 and 14 days following treatment,and at various storage temperatures, e.g. room temperature and 56° C.The results are provided in FIG. 3.

B. Wicking Time

The wicking time of cystine treated gold and palladium test strips wasdetermined as described in Procedure C found in Appendix A, infra. Thewicking time was determined at various times following surfacetreatment, i.e. 0, 7 and 14 days following treatment, and at variousstorage temperatures, e.g. room temperature and 56° C. The results areprovided in FIG. 4.

III. Characterization of MESA Treated Electrochemical Test Strips

A. Contact Angle

The contact angle of MESA treated gold and palladium test strips(treated at pH 5.4 and 11.5) was determined with water and a goniometeras described in Procedure B found in Appendix A, infra. The contactangle was determined at various times following surface treatment, i.e.0, 7 and 14 days following treatment where the storage temperature was56° C. The results are provided in FIGS. 5A (pH 5.4) and 5B (pH 11.5).

B. Wicking Time

The wicking time of MESA treated gold and palladium test strips (treatedat pH 5.4 and 11.5) was determined as described in Procedure B found inAppendix A, infra. The wicking time was determined at various timesfollowing surface treatment, i.e. 0, 7 and 15 days following treatment,and at various storage temperatures, e.g. room temperature and 56° C.The results are provided in FIG. 6.

IV. Wicking Time Comparison Study

The wicking time of three different electrochemical test strips preparedas described above was compared. The first electrochemical test strip(Case A) was one in which both the gold and palladium surfaces werecystine treated. The second electrochemical test strip (Case B) was onein which both the palladium and gold surfaces were treated with MESA.The third electrochemical test strip (Case C) was one in which thepalladium surface was cystine treated and the gold surface was MESAtreated. Wicking times were determined as described in Procedure C foundin Appendix A, infra, on strips stored in SureStep® vials at 56° C. for7 and 14 days, and the results are provided in FIG. 7.

The above results and discussion demonstrate that significantly improvedelectrochemical test strips for use in the determination of an analytein a test sample are provided by the subject invention. Specifically,storage stable electrochemical test strips having durable hydrophilicsurfaces that exhibit low interference to the electrochemicalmeasurement of oxidized species and have fast wicking times areprovided. Furthermore, the surface modifying reagents used to modify thesurfaces of the subject test strips are odorless. As such, the subjectinvention represents a significant contribution to the art.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference. The citation of any publication is for its disclosure priorto the filing date and should not be construed as an admission that thepresent invention is not entitled to antedate such publication by virtueof prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Appendix A Procedure A

Surface Treatment Procedure for Gold and Palladium Metallized Plastics

Materials:

-   -   1. Pyrex glass baking dish size 4 Q (10.5″×14.75″×2.25″)    -   2. Mill-Q Water    -   3. Stop watch    -   4. Gold and Palladium sheets size 12″8.5″        Chemical:    -   2-mercaptoethane sulfonic acid, sodium salt    -   Manufacturer TCI    -   Catalog # M0913    -   Purity: 99%    -   Molecular Wt. 164.18        Procedure:    -   0.1% (w/w) MESA    -   1. Weigh out 1.000(±0.0005) g of 2-mercaptoethane sulfonic acid        sodium in a weighing paper.    -   2. Weigh out 999.0(±0.11) g of Milli Q water in a glass beaker.    -   3. Add MESA powder slowly to the beaker. Let it dissolve        completely    -   Surface Treatment:    -   4. Cut out Gold and Palladium sheets (size 12″×8.5″) from the        roll.    -   5. Pour out the content of beaker to the baking dish slowly.    -   6. Coat metal sheets one by one, metal layer facing dish bottom.        Make sure sheet is completely covered with solution. Use the        stopwatch to monitor coating time (1 min/sheet).    -   7. Drying time is about 1 hr.    -   8. Check the contact angle of Metallized film with water by        Goniometer. Contact angle should be <20° for Au and Pd surfaces.

Procedure B

Contact Angle Measurement Using Rame-Hart Goniometer

Materials:

-   -   1. MESA coated Gold and Palladium sheet    -   2. Rame-Hart Goniometer Model-100-00-115    -   3. Automated Pipetting system    -   4. Software RHI 2001        Procedure: Using water, fill up the Automated Pipette system.        Place the sample (Au/Pd) on the sample platform and hold with        clamp. Open RHI 2001 program and set up the baseline. Drop 3 to        5 uL of water from automatic pipette. RHI 2001 system captures        the image and measure the contact angle from both sides and        averages them. This can be repeated for several times.

Procedure C

Wicking Time Measurement

Material:

-   -   1. MESA treated test strips    -   2. Fresh blood adjusted to 70% Hematocrit    -   3. Pipette—20 uL    -   4. Pieces of Parafilm for blood application.    -   5. Panasonic camera model GP KP222    -   6. Adobe Premiere software 4.2 for video capture    -   7. Computer System and a Monitor    -   8. Two side adhesive tape & a platform for strip        Procedure:    -   1. Place a strip on a platform and hold it with tape.    -   2. Place the strip under the camera lens and adjust the focus        and magnification.    -   3. Launch the Premiere software and open movie captures program.        Select 30 fps NTSC system for capturing live movie.    -   4. Place 5 uL of 70% hot blood on Parafilm surface.    -   5. Turn on recording mode and apply blood from either side of        test strip in to the capillary.    -   6. Turn off the recording mode when blood reaches the other end        of test strip    -   7. Go to the image window and analyze it. Use In mark when blood        touches the strip and out mark when blood reaches the other end.        Software does the frames count (30 frames/seconds) and displays        in lower window.    -   8. To calculate wicking time, divide number of frames with 30.    -   9. Repeat the procedure for # of strips

1. A kit for use in determining the concentration of an analyte in aphysiological sample, said kit comprising: (a) an electrochemical teststrip comprising: (i) a reaction zone defined by opposing working andreference metallic electrodes separated by a spacer layer, wherein atleast one of said first and second metallic electrodes has a surfacemodified with a homogenous surface modification layer made up ofidentical self assembling molecules having a first sulfhydryl end groupand a second sulfonate end group, wherein said sulfhydryl and sulfonateend groups are separated by a lower alkyl linker group and wherein thehomogeneous surface modification layer renders the surface of the atleast one of said first and second metallic electrodes stablyhydrophilic; and (ii) a redox reagent system present in said reactionzone, wherein said redox reagent system comprises at least one enzymeand a mediator; and (b) at least one of: (i) a means for obtaining saidphysiological sample; and  an analyte standard.
 2. The kit according toclaim 1, wherein said analyte is glucose.
 3. The kit according to claim1, wherein said physiological sample is blood.
 4. The kit according toclaim 1, wherein said means for obtaining said physiological sample is alance.
 5. The kit according to claim 1, wherein said kit furthercomprises an automated instrument for detecting an electrical signalusing said electrodes and relating said detected signal to the amount ofanalyte in a sample.
 6. A system for use in determining theconcentration of an analyte in a physiological sample, said systemcomprising: (a) an electrochemical test strip comprising: (1) a reactionzone defined by opposing working and reference metallic electrodesseparated by a spacer layer, wherein at least one of said first andsecond metallic electrodes has a surface modified with a homogenoussurface modification layer made up of identical self assemblingmolecules having a first sulfhydryl end group and a second sulfonate endgroup, wherein, said sulfhydryl and sulfonate end groups are separatedby a lower alkyl linker group and wherein the homogeneous surfacemodification layer renders the surface of the at least one of said firstand second metallic electrodes stably hydrophilic; and (2) a redoxreagent system present in said reaction zone, wherein said redox reagentsystem comprises at least one enzyme and a mediator; and (b) anautomated instrument.